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J. Nat. Prod. 2001, 64, 1052-1055
Isolation of New Protein Phosphatase Inhibitors from Two Cyanobacteria Species, Planktothrix spp. Tomoharu Sano,*,† Takeo Usui,‡ Kazunori Ueda,‡,§ Hiroyuki Osada,‡ and Kunimitsu Kaya† National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan, RIKEN, 2-1 Hirosawa, Wako, Saitama 305-0198, Japan, and Toyama Prefectural University, 5180 Kurokawa, Kosugi, Imizu, Toyama 939-0398, Japan Received November 17, 2000
Two new protein phosphatase inhibitors, oscillamide B (1) and C (2), were isolated from the cyanobacteria Planktothrix (Oscillatoria) agardhii and P. rubescens. The structures of the inhibitors were elucidated by analysis of HRFABMS, 1D and 2D NMR spectra, and chemical degradation. These inhibitors are ureido-containing cyclic peptides and inhibited serine/threonine protein phosphatases PP1 and PP2A. The inhibitory activities were closely related to the Arg and N-Me-Hty residues in the peptides. The toxic freshwater cyanobacteria (blue-green algae) Planktothrix agardhii (Gomont) Anagnostidis & Koma´rek (formerly Oscillatoria agardhii Gomont) (Oscillatoriales, Oscillatoriaceae) and Planktothrix rubescens (DC. ex Gomont) Anagnostidis & Koma´rek (formerly Oscillatoria rubescens DC. ex Gomont) (Oscillatoriales, Oscillatoriaceae) produce many kinds of bioactive cyclic peptides.1 Microcystins, hepatotoxic cyclic heptapeptides from the cyanobacteria, have been found to inhibit protein phosphatase type 1 and 2A (PP1 and PP2A).2 Dehydrobutyrine-containing microcystins also hepatotoxic cyclic peptides, and a protein phosphatase-inhibiting cyclic peptide has also been isolated from the filamentous cyanobacteria Nostoc and Planktothrix.3-5 Specific inhibitors of protein phosphatases can be utilized to reveal the role of each protein phosphatase that controls the phosphorylation level of proteins. The control of protein phosphorylation level is very important for regulating intracellular events in eukaryotic cells.6 During our investigation of peptides in P. agardhii and P. rubescens, we found two novel protein phosphatase-inhibiting cyclic peptides, 1 and 2. In this paper, we report the structure elucidation of 1 and 2 and their structureactivity relationship as protein phosphatase inhibitors. Results and Discussion The extract from P. agardhii (CCAP 1459/11A), prepared by soaking with 5% acetic acid aqueous solution, showed inhibitory activity against PP1 and PP2A. The extract was fractionated using Sep-Pak C18 cartridges with 20% and 80% aqueous MeOH. The 80% MeOH eluent was chromatographed on reversed-phase HPLC (65% MeOH in 50 mM phosphate buffer, pH 3.0). The active fraction was further purified by normal phase HPTLC to give 21.0 mg (0.2% yield from dry algae) of oscillamide B (1). Oscillamide B (1) was a colorless, amorphous solid. The molecular formula of 1 was established as C41H60O9N10S from the positive high-resolution FABMS spectrum ([M + H]+ at m/z 869.4324, calcd for C41H61O9N10S, 869.4343). The 1H and 13C NMR spectral data of 1 (Table 1) suggested that it is a peptide. After acid hydrolysis of 1 (6 M HCl, 150 °C, 10 h), homotyrosine (Hty), arginine (Arg), phenylalanine (Phe), * To whom correspondence should be addressed. Tel: +81-298-50-2449. Fax: +81-298-50-2574. E-mail:
[email protected]. † National Institute for Environmental Studies. ‡ RIKEN. § Toyama Prefectural University.
10.1021/np0005356 CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 08/03/2001
New Protein Phosphatase Inhibitors Table 1. 1H and in CD3OD
13C
NMR Spectral Data for Oscillamide B (1)
1H
position Phe
N-Me-Ala
Hty
1 2 3 4 5, 9 6, 8 7 1 2 3 N-Me 1 2 3 4
Met
Lys
5 6, 10 7, 9 8 1 2 3 4 5 1 2 3 4 5 6
Arg
1 2 3
Journal of Natural Products, 2001, Vol. 64, No. 8 1053
4.59 3.38 2.82 7.10 7.24 7.15 4.72 1.14 1.85 4.74 2.05 1.79 2.73 2.57
J (Hz)
(dd, 3.4, 12.8) (dd, 3.4, 12.8) (t, 12.8) (d, 7.3) (t, 7.3) (t, 7.3) (q, 6.7) (d, 6.7) (s) (dd, 5.2, 9.2) (m) (m) (m) (m)
7.06 6.71
(d, 8.2) (d, 8.2)
4.33 2.13 2.68 2.13
(t, 7.6) (m) (m) (s)
4.12 1.90 1.70 1.50 1.34 1.56 3.70 2.93
(dd, 4.3, 10.1) (m) (m) (m) (m) (m) (m) (m)
4.14 1.85 1.67 1.63 3.20
(dd, 4.4, 6.0) (m) (m) (m) (m)
4 5 6 CO (ureido)
HMBC (CfH)
13C
174.1 56.8 39.1
2, 3, Lys-6
139.0 130.1 129.9 127.7 172.2 56.5 14.2 28.2 174.1
3 3
Table 2. 1H and in CD3OD
132.7 130.5 116.4 157.0 175.1 54.8 32.0 31.2 15.5 176.4 56.3 32.8
Phe
2 3 4
2, 3, N-MeAla-2, N-Me
6, 10 Hty
2, 3, Hty-2 4 5 4 2, 3, Met-2 Ile
29.4 40.0
26.1 42.1 158.6 159.8
5 6, 10 7, 9 8 N-Me 1 2 3
21.7
179.7 55.6 32.0
1 2 3
4 5, 9 6, 8 7 N-Me-Hty 1
2, 3, Phe-2 N-Me
4
2, 3
Lys
5 6, 10 7, 9 8 1 2 3 4 5 6 1 2 3 4 5 6
Arg-2
lysine (Lys), methionine (Met), and N-methylalanine (NMe-Ala) were detected by amino acid analysis using Marfey’s method7 and a chiral GC/MS method.8 From the amino acid analysis, Lys was identified as being in the D-configuration, and all other amino acids are in the L-configuration. From the amino acid composition and the molecular formula, the structure of 1 was expected to be a Met variant of anabaenopeptin F (3)9 at the isoleucine (Ile) position. From extensive analysis of the 2D NMR spectra, such as COSY, HOHAHA, HMQC, and HMBC, these amino acid units were confirmed. The amino acid sequences of 1 were determined from the HMBC spectrum. The crosspeak between H-6 of Lys and the carbonyl carbon of Phe confirmed the connectivity. The connectivity between NMe-Ala and Hty was suggested by the cross-peak between the N-Me proton of N-Me-Ala and the carbonyl carbon of Hty. The other connectivities were confirmed with the cross-peaks between the R-protons and the carbonyl carbons. These results established the structure of oscillamide B as 1. Following the same procedure as used for 1, oscillamide C (2, 7.7 mg, 0.1% yield from dry algae) was isolated as a colorless solid from P. rubescens (CCAP 1459/14). The molecular formula of 2 was established as C49H68O10N10
NMR Spectral Data for Oscillamide C (2) 1H
position
50.2 34.7 32.1
13C
Arg
1 2 3 4 5
J (Hz)
4.62 3.38 2.87
(dd, 3.4, 12.5) (dd, 3.4, 14.0) (dd, 12.8,14.0)
7.10 7.22 7.15
(d, 7.3) (t, 7.3) (t, 7.3)
4.47 2.01 1.60 2.32 2.21
(dd, 5.8, 8.6) (m) (m) (m) (m)
6.92 6.67
(d, 8.5) (d, 8.5)
1.89
(s)
4.67 2.07 1.81 2.78 2.66
(dd, 3.7, 10.7) (m) (m) (m) (m)
7.06 6.70
(d, 8.6) (d, 8.6)
4.06 1.92 1.78 1.28 1.01 1.00
(t, 9.2) (m) (m) (m) (t, 7.3) (d, 7.0)
4.12 1.95 1.75 1.55 1.35 1.62 3.70 3.00
(dd, 4.3, 5.5) (m) (m) (m) (m) (m) (m) (m)
4.15 1.85 1.67 1.65 3.23 3.18
(dd, 6.4, 7.0) (m) (m) (m) (m) (m)
6 CO (ureido)
13C
HMBC (CfH)
174.1 56.8 38.9
2, 3, Lys-6
139.1 130.1 129.7 127.6 171.7
3 3 2, 3, N-Me, Phe-2
61.1 31.4 33.0 132.8 130.0 116.4 157.1 28.3 174.4
4 4
2, N-Me-Hty2, N-Me
50.0 34.6 31.9 132.5 130.8 116.5 157.5 175.1 60.1 37.5 26.7 12.0 16.3 176.2 56.5 32.5
4 4 2
2, 3, Ile-2
21.8 29.3 40.2 179.4 55.5 32.2
2, 3
26.0 42.1 158.6 159.7
5 Lys-2, Arg-2
on the basis of high-resolution FABMS data ([M + H]+ at m/z 957.5198, calcd for C49H69O10N10, 957.5198). The 1H and 13C NMR spectral data of 2 (Table 2) suggested that 2 was a peptide. The amino acids Hty, Arg, Phe, Lys, Ile, and N-methylhomotyrosine (N-Me-Hty) were detected by Marfey’s method and chiral GC/MS analysis after acid hydrolysis (6 M HCl, 150 °C, 10 h). The stereochemistry of these amino acids were elucidated. The configuration of Lys was shown to be D, and all other amino acids were identified as having the L-configuration. This amino acid composition and the molecular formula suggested that 2 was the N-Me-Hty variant of 3 at the N-Me-Ala position. The amino acid sequences of 2 were determined from HMBC and ROESY spectra. From these data, the structure of oscillamide C was confirmed as 2.
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Journal of Natural Products, 2001, Vol. 64, No. 8
Sano et al.
1-4 the N-methyl amino acid unit was located at Z.4 When Z4 was N-Me-Hty as in peptide 2, the inhibitory activities against PP1 and PP2A were much greater than when Z4 is N-Me-Ala. The amino acid unit at Y2 did not significantly affect the inhibitory activities. From these findings, the structure-activity relationship suggests that N-Me-Hty at Z4 and a ureido-linked Arg at X in the peptides are closely related to the inhibition of PP1 and PP2A activities, but N-Me-Ala at Z4 and ureido-linked Tyr at X are not. A similar structure-activity relationship was observed in the case of inhibition of carboxypeptidase A by anabaenopeptins.11 The positive charge of the guanidyl group of Arg and the hydrophobic residue of N-Me-Hty in the peptides may play an important role in the inhibition of PP1 and PP2A. The reversible phosphorylation of proteins is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells.6 Protein phosphatases act as key enzymes in the control of the phosphorylation level of proteins. Therefore, these protein phosphatase inhibitors can be utilized for biological research. Experimental Section
Figure 1. (A) Inhibitory activities of 1-4 against protein phosphatases. (B) Inhibition of PP1 and PP2A by 2.
Table 3. Structures and Protein Phosphatases Inhibition Activities of Peptides 1-4 inhibition (%) at 100 µg/mL Y2
peptide 1 2 3 4
Lys Lys Lys Lys
Met Ile Ile Ile
Z4 Hty Hty Hty Hty
N-Me-Ala N-Me-Hty N-Me-Ala N-Me-Ala
Phe Phe Phe Phe
X
PP1
PP2A
Arg Arg Arg Tyr
43.3 97.3 38.1 11.2
62.8 98.4 41.5 9.6
Another inhibitor (3) was isolated from P. rubescens (NIES-610), and its structure was elucidated in the same way as 1 and 2. From the molecular formula, amino acid composition, and 1D and 2D NMR spectra, 3 was suggested to be identical with anabaenopeptin F. The inhibitory activities of these cyclic peptides (1-3) and oscillamide Y (4)10 against protein phosphatases were tested. The peptides showed inhibitory activities against protein serine/threonine phosphatase PP1 and PP2A at 100 µg/mL (Figure 1), except for peptide 4, but did not inhibit protein tyrosine phosphatase (PTP-S2) or dual-specificity phosphatase (VHR and Cdc25B) activity even at 100 µg/ mL (Figure 1). The most effective inhibitor of the peptides tested was peptide 2, which had IC50 values of 0.90 and 1.33 µM against PP1 and PP2A, respectively. The inhibitory activity values (%) of 2 against PP1 and PP2A at 100 µg/mL were significantly different from those of 1 by the Student’s t-test (R ) 0.001) after arcsine transformation. The general structure of the tested peptides was shown to be cyclo[(X-R-NH-CO-R-NH)-Lys1-Y2-Hty3-Z4-Phe5--NH(Lys1)]. In the case of peptide 1, X, Y2, and Z4 were Arg, Met, and N-Me-Ala, respectively (Table 3). Also, in peptide 2, X, Y2, and Z4 were Arg, Ile, and N-Me-Hty, and X, Y2, and Z4 of peptide 3 were Arg, Ile, and N-Me-Ala, respectively. In peptide 4, X, Y2, and Z4 were shown to be Tyr, Ile, and N-Me-Ala, respectively. When X was Arg as in peptides 1, 2, and 3, considerable inhibitory activities against PP1 and PP2A were observed, but in the case of Tyr, no inhibition of the enzymes was observed. In peptides
General Procedures. NMR spectra were recorded on a JEOL JNM A-500 spectrometer (500 MHz for 1H and 125 MHz for 13C) in CD3OD. 1H and 13C chemical shifts are referenced to the solvent peaks (1H, 3.30 ppm, and 13C, 49.5 ppm in CD3OD). Low- and high-resolution FABMS spectra were performed with a JEOL JMS-700 spectrometer using glycerol as the matrix. Specific rotations were obtained on an Atago POLAX-D polarimeter. Chemicals. D- and L-homotyrosine were gifts from Dr. Mark Bradley at University of Southampton, U.K. All other solvents and reagents were purchased from commercial sources. Culture Conditions. Planktothrix agardhii and P. rubescens (CCAP 1459/11A and CCAP 1459/14) were kindly provided by Dr. John G. Day of CCAP, Scotland. P. rubescens (NIES-610 ) CCAP 1459/22 ) NIVA CYA 18) was obtained from National Institute for Environmental Studies Microbial Culture Collection. Planktothrix were grown in 10 L culture bottles with CT medium [Ca(NO3)2‚4H2O (15 mg), KNO3 (10 mg), β-Na2 glycerophosphate (5 mg), MgSO4‚7H2O (4 mg), vitamin B12 (0.01 µg), biotin (0.01 µg), thiamin HCl (1 µg), FeCl3‚6H2O (0.06 mg), MnCl2‚4H2O (0.01 mg), ZnSO4‚7H2O (7 µg), CoCl2‚6H2O (1.2 µg), Na2MoO4‚2H2O (0.75 µg), Na2EDTA‚ 2H2O (0.3 mg), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) (40 mg), distilled water 100 mL, pH 8.2].12 The cells were grown isothermally at 20 °C (light intensity, below 250 µmol photon m-2 s-1; aeration rate, 1.5 L min-1) for 3 weeks. The cultured cells were harvested by continuous flow centrifugation at 10 000 rpm. The harvested cells were lyophilized and kept in a freezer at -20 °C until extraction. Extraction and Isolation. The freeze-dried algae were suspended in 5% aqueous acetic acid solution. The suspensions were centrifuged (3500g, 10 min), and the supernatants were passed through Sep-Pak C18 cartridges (Waters). The cartridges were washed with 20% aqueous MeOH and eluted with 80% aqueous MeOH. The eluate was evaporated in vacuo. The residue was dissolved in 20% aqueous MeOH and separated by HPLC (column, Mightysil RP-18 20 × 250 mm (Kanto Chemical Co., Inc.); eluent, 65% MeOH in 50 mM phosphate buffer, pH 3.0; flow rate, 9 mL/min). The active fractions were further purified with HPTLC (Si gel 60 F254 (Merck), thickness, 0.25 mm; developing solvent, CHCl3-MeOH-H2O (6:4: 1)). Oscillamide B (1, tR 9.3 min, Rf 0.7) and oscillamide C (2, tR 8.3 min, Rf 0.8) were obtained from CCAP 1459/11A and CCAP 1459/14, respectively. Compound 3 (anabaenopeptin F) was isolated from P. rubescens (NIES-610) in the same way as 1 and 2. Compound 4 (oscillamide Y) was isolated previously.10
New Protein Phosphatase Inhibitors
Protein Phosphatase Inhibition Assay. Protein phosphatase type-1 (PP1 from rabbit skeletal muscle) and protein phosphatase type-2A (PP2A from human red blood cells) were purchased from Upstate Biotechnology (NY). In vitro protein phosphatase assays were carried out in duplicate at 30 °C for 10 min using 0.05 unit/50 µL of protein phosphatase and 32Plabeled myelin basic protein (New England Biolabs), which was prepared from the catalytic subunit of cAMP-dependent kinase and γ-[32P]ATP. The reaction was terminated by addition to each sample of 200 µL of 20% trichloroacetic acid and chilling on ice. After centrifugation, the clear supernatant was collected and the amount of the radioactivity released as 32Pi was determined using a liquid scintillation counter (Packard TRICURVE 2000). Control experiments were performed by addition of MeOH. Tyrosine phosphatase PTP-S2 and dual-specificity phosphatase VHR13 were overexpressed in Escherichia coli JM109, and phosphatase assays were performed in triplicate according to our previous report.14 The catalytic subunit of human Cdc25B was also expressed in BL21 (DE3) using pET21b (Novagen) and purified according to the methods of Fauman et al.15 Phosphatase assays were performed at 37 °C using 200 µM 3-O-methylfluorescein phosphate as a substrate in 0.7 mL of reaction solution (100 mM Tris-HCl, 40 mM NaCl, 1 mM dithiothreitol, 20% glycerol, pH8.2). After incubation for 20 min, the reaction was terminated by adding 0.55 mL of the stop solution (6 M guanidine-HCl, 1 M 2-(N-morpholino)ethanesulfonic acid, pH 5.5), and the increase in the absorbance at 477 nm was measured. Control experiments were performed by addition of MeOH. Oscillamide B (1): colorless solid (21.0 mg, 0.2% dry wt); [R]25D -83° (c 0.2, MeOH); UV (MeOH) λmax (log ) 279 nm (3.5); 1H and 13C NMR, see Table 1; FABMS (pos. glycerol) m/z 869 [M + H]+; HRFABMS (pos., glycerol) m/z 869.4324 [M + H]+ (calcd for C41H61O9N10S, 869.4343). Oscillamide C (2): colorless solid (7.7 mg, 0.1% dry wt); [R]25D -111° (c 0.15, MeOH); UV (MeOH) λmax (log ) 279 nm
Journal of Natural Products, 2001, Vol. 64, No. 8 1055
(3.5); 1H and 13C NMR, see Table 2; FABMS (pos. glycerol) m/z 957 [M + H]+; HRFABMS (pos., glycerol) m/z 957.5198 [M + H]+ (calcd for C49H69O10N10, 957.5198). Anabaenopeptin F (3): colorless solid (25.2 mg, 0.03% dry wt); [R]25D -60° (c 0.08, MeOH) (lit. -41.4°, c 0.1, MeOH); UV (MeOH) λmax (log ) 279 nm (3.2); FABMS (pos. glycerol) m/z 851 [M + H]+; HRFABMS (pos., glycerol) m/z 851.4818 [M + H]+ (calcd for C42H63O9N10, 851.4779). Acknowledgment. The authors thank Drs. H. Ito and S. Serizawa (NIES) for MS analyses. References and Notes (1) Moore, R. E. J. Ind. Microbiol. 1996, 16, 134-143. (2) Yoshizawa, S.; Matsushima, R.; Watanabe, M. F.; Harada, K.; Ichihara, A.; Carmichael, W. W.; Fujiki, H. J. Cancer Res. Clin. Oncol. 1990, 116, 609-614. (3) Sano, T.; Kaya, K. Tetrahedron 1998, 54, 463-470. (4) Beattie, K. A.; Kaya, K.; Sano, T.; Codd, G. A. Phytochemistry 1998, 47, 1289-1292. (5) Kaya, K.; Sano, T.; Beattie, K. A.; Codd, G. A. Tetrahedron Lett. 1996, 37, 6725-6728. (6) Cohen, P. Annu. Rev. Biochem. 1989, 58, 453-508. (7) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596. (8) Sano, T.; Kaya, K. J. Nat. Prod. 1996, 59, 90-92. (9) Shin, H. J.; Matsuda, H.; Murakami, M.; Yamaguchi, K. J. Nat. Prod. 1997, 60, 139-141. (10) Sano, T.; Kaya, K. Tetrahedron Lett. 1995, 36, 5933-5936. (11) Itou, Y.; Suzuki, S.; Ishida, K.; Murakami, M. Bioorg. Medic. Chem. Lett. 1999, 9, 1243-1246. (12) Watanabe, M. M.; Hiroki, M. Eds. NIES-Collection. List of Strains; National Institute for Environmental Studies: Tsukuba, Japan, 1997; p 31. (13) Ishibashi, T.; Bottaro, D. P.; Chan, A.; Miki, T.; Aaronson, S. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 12170-12174 (14) Usui, T.; Marriott, G.; Inagaki, M.; Swarup, G.; Osada, H. J. Biochem. 1999, 125, 960-965. (15) Fauman, E. B.; Cogswell, J. P.; Lovejoy, B.; Rocque, W. J.; Holmes, W.; Montana, V. G.; Piwnica-Worms, H.; Rink, M. J.; Saper, M. A. Cell 1998, 93, 617-625.
NP0005356
